Creation and preclinical evaluation of a novel mussel-inspired, biomimetic, bioactive bone graft scaffold: direct comparison with Infuse bone graft using a rat model of spinal fusion

Ethan Cottrill Departments of Neurosurgery,
Department of Orthopaedic Surgery, Duke University Health System, Durham, North Carolina;

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Zach Pennington Departments of Neurosurgery,
Department of Neurologic Surgery, Mayo Clinic, Rochester, Minnesota;

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Matthew T. Wolf Biomedical Engineering, and
Laboratory of Cancer Immunometabolism, Center for Cancer Research, National Cancer Institute, Frederick, Maryland;

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Naomi Dirckx Orthopaedic Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland;

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Jeff Ehresman Departments of Neurosurgery,
Department of Neurosurgery, Barrow Neurological Institute, Phoenix, Arizona;

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Alexander Perdomo-Pantoja Departments of Neurosurgery,

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Christian Rajkovic Departments of Neurosurgery,

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Jessica Lin Departments of Neurosurgery,

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David R. Maestas Jr. Biomedical Engineering, and

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Ashlie Mageau Biomedical Engineering, and

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Dennis Lambrechts Biomedical Engineering, and

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Veronica Stewart Departments of Chemistry and
Materials Science and Engineering, Johns Hopkins University, Baltimore, Maryland; and

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Daniel M. Sciubba Departments of Neurosurgery,
Department of Neurosurgery, Donald and Barbara Zucker School of Medicine at Hofstra/Northwell Health, Hempstead, New York

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Nicholas Theodore Departments of Neurosurgery,

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Jennifer H. Elisseeff Biomedical Engineering, and

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Timothy Witham Departments of Neurosurgery,

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OBJECTIVE

Infuse bone graft is a widely used osteoinductive adjuvant; however, the simple collagen sponge scaffold used in the implant has minimal inherent osteoinductive properties and poorly controls the delivery of the adsorbed recombinant human bone morphogenetic protein–2 (rhBMP-2). In this study, the authors sought to create a novel bone graft substitute material that overcomes the limitations of Infuse and compare the ability of this material with that of Infuse to facilitate union following spine surgery in a clinically translatable rat model of spinal fusion.

METHODS

The authors created a polydopamine (PDA)–infused, porous, homogeneously dispersed solid mixture of extracellular matrix and calcium phosphates (BioMim-PDA) and then compared the efficacy of this material directly with Infuse in the setting of different concentrations of rhBMP-2 using a rat model of spinal fusion. Sixty male Sprague Dawley rats were randomly assigned to each of six equal groups: 1) collagen + 0.2 µg rhBMP-2/side, 2) BioMim-PDA + 0.2 µg rhBMP-2/side, 3) collagen + 2.0 µg rhBMP-2/side, 4) BioMim-PDA + 2.0 μg rhBMP-2/side, 5) collagen + 20 µg rhBMP-2/side, and 6) BioMim-PDA + 20 µg rhBMP-2/side. All animals underwent posterolateral intertransverse process fusion at L4–5 using the assigned bone graft. Animals were euthanized 8 weeks postoperatively, and their lumbar spines were analyzed via microcomputed tomography (µCT) and histology. Spinal fusion was defined as continuous bridging bone bilaterally across the fusion site evaluated via µCT.

RESULTS

The fusion rate was 100% in all groups except group 1 (70%) and group 4 (90%). Use of BioMim-PDA with 0.2 µg rhBMP-2 led to significantly greater results for bone volume (BV), percentage BV, and trabecular number, as well as significantly smaller trabecular separation, compared with the use of the collagen sponge with 2.0 µg rhBMP-2. The same results were observed when the use of BioMim-PDA with 2.0 µg rhBMP-2 was compared with the use of the collagen sponge with 20 µg rhBMP-2.

CONCLUSIONS

Implantation of rhBMP-2–adsorbed BioMim-PDA scaffolds resulted in BV and bone quality superior to that afforded by treatment with rhBMP-2 concentrations 10-fold higher implanted on a conventional collagen sponge. Using BioMim-PDA (vs a collagen sponge) for rhBMP-2 delivery could significantly lower the amount of rhBMP-2 required for successful bone grafting clinically, improving device safety and decreasing costs.

ABBREVIATIONS

BV = bone volume; ECM = extracellular matrix; HSD = honestly significant difference; PDA = polydopamine; rhBMP-2 = recombinant human bone morphogenetic protein–2; SEM = scanning electron microscopy; TN = trabecular number; TS = trabecular separation; μCT = microcomputed tomography.

OBJECTIVE

Infuse bone graft is a widely used osteoinductive adjuvant; however, the simple collagen sponge scaffold used in the implant has minimal inherent osteoinductive properties and poorly controls the delivery of the adsorbed recombinant human bone morphogenetic protein–2 (rhBMP-2). In this study, the authors sought to create a novel bone graft substitute material that overcomes the limitations of Infuse and compare the ability of this material with that of Infuse to facilitate union following spine surgery in a clinically translatable rat model of spinal fusion.

METHODS

The authors created a polydopamine (PDA)–infused, porous, homogeneously dispersed solid mixture of extracellular matrix and calcium phosphates (BioMim-PDA) and then compared the efficacy of this material directly with Infuse in the setting of different concentrations of rhBMP-2 using a rat model of spinal fusion. Sixty male Sprague Dawley rats were randomly assigned to each of six equal groups: 1) collagen + 0.2 µg rhBMP-2/side, 2) BioMim-PDA + 0.2 µg rhBMP-2/side, 3) collagen + 2.0 µg rhBMP-2/side, 4) BioMim-PDA + 2.0 μg rhBMP-2/side, 5) collagen + 20 µg rhBMP-2/side, and 6) BioMim-PDA + 20 µg rhBMP-2/side. All animals underwent posterolateral intertransverse process fusion at L4–5 using the assigned bone graft. Animals were euthanized 8 weeks postoperatively, and their lumbar spines were analyzed via microcomputed tomography (µCT) and histology. Spinal fusion was defined as continuous bridging bone bilaterally across the fusion site evaluated via µCT.

RESULTS

The fusion rate was 100% in all groups except group 1 (70%) and group 4 (90%). Use of BioMim-PDA with 0.2 µg rhBMP-2 led to significantly greater results for bone volume (BV), percentage BV, and trabecular number, as well as significantly smaller trabecular separation, compared with the use of the collagen sponge with 2.0 µg rhBMP-2. The same results were observed when the use of BioMim-PDA with 2.0 µg rhBMP-2 was compared with the use of the collagen sponge with 20 µg rhBMP-2.

CONCLUSIONS

Implantation of rhBMP-2–adsorbed BioMim-PDA scaffolds resulted in BV and bone quality superior to that afforded by treatment with rhBMP-2 concentrations 10-fold higher implanted on a conventional collagen sponge. Using BioMim-PDA (vs a collagen sponge) for rhBMP-2 delivery could significantly lower the amount of rhBMP-2 required for successful bone grafting clinically, improving device safety and decreasing costs.

In Brief

The authors created a novel bone graft substitute material and used a rat model of spinal fusion to compare the efficacy of this new material against that of the widely used Infuse bone graft product in the setting of different concentrations of recombinant human bone morphogenetic protein-2 (rhBMP-2). The new material resulted in complete fusions with bone volume and quality superior to that seen with the use of Infuse collagen sponges with a 10-fold higher concentration of rhBMP-2. Additional work is needed to evaluate this new material more fully as a bone graft substitute.

Over 500,000 patients undergo thoracolumbar fusion annually,1,2 accounting for the highest aggregate hospital cost of any inpatient procedure, estimated at more than $12 billion annually.3 Long-term clinical improvements following instrumented fusion have been correlated with successful radiographic evidence of fusion.4,5 For that reason, there is great interest among patients, payors, and providers to optimize the likelihood of osseous union at the fusion site. Nevertheless, nonunion of fusion continues to occur in 15%–20% of patients,6,7 which has led to the development of many adjuvant technologies,8,9 including novel bone graft substitutes,1013 to reduce the occurrence of this complication.

Among bone graft substitute materials, Infuse bone graft (Medtronic) is among the most widely used because of its demonstrable clinical efficacy.14,15 The Infuse graft, which comprises a solution of recombinant human bone morphogenetic protein–2 (rhBMP-2) that is applied topically to a collagen sponge, has been widely successful since its FDA approval in 2002. However, despite the high fusion rates seen with Infuse-supplemented fusions, the product has been associated with serious complications, including inflammatory reactions, radiculopathy, osteolysis, and urogenital disorders.16,17 These complications have been hypothesized to stem from the noncontrolled burst release of the enormous amounts of rhBMP-2 required for fusion, which exceed physiological levels by more than 106-fold.1820 We explored the opportunity to avoid complications while still providing high rates of fusion by developing a new rhBMP-2 delivery scaffold with superior inherent osteoinductivity and a drug delivery process with fewer adverse physiological effects.

Here, we report the creation of such a scaffold (Table 1) and our comparison of its ability with that of Infuse for promoting radiographic union following spine surgery in a clinically translatable rat model of spinal fusion. For rhBMP-2 delivery, we utilized polydopamine (PDA), a natural adhesive inspired by the substances produced by mussels for adhering to underwater surfaces, which confers the theoretical advantage of controlled and sustained growth factor release.2123

TABLE 1.

Identified limitations of the Infuse bone graft and design solutions provided in BioMim-PDA

Infuse Bone Graft LimitationDesign Solution in BioMim-PDAAdvantage of Design Solution
Simple collagen sponge used alone as a bone graft substitute creates no boneMake a biomimetic bone graft* that creates bone on its own & retains the handleability of a collagen spongeBiomimetic bone grafts (e.g., collagens combined w/ calcium phosphates) create bone on their own, which may lead to less rhBMP-2 needed for fusion
Use pro-regenerative ECM instead of collagen aloneECMs comprise many pro-regenerative components, e.g., collagens, proteoglycans, glycosaminoglycans, glycoproteins, & growth factors, & may be superior to collagen alone as sources of organic components for bone formation
Nonphysiologic, uncontrolled growth factor deliveryUse a mussel-inspired growth factor delivery strategy (PDA)Controlled, sustained growth factor delivery may avoid the initial "burst release" of growth factors & side effects seen w/ Infuse & lead to enhanced bone formation at reduced dosages of growth factor & reduced costs

Biomimetic bone graft is a material similar in composition to bone, i.e., has both organic (e.g., collagen) and inorganic (e.g., calcium phosphate) components.

Methods

Creation of a Novel Mussel-Inspired, Biomimetic, Bioactive Bone Graft Substitute Material

We created a novel mussel-inspired, biomimetic, bioactive bone graft substitute material, BioMim-PDA—a porous, homogeneously dispersed solid mixture of pro-regenerative extracellular matrix (ECM) and calcium phosphates infused with PDA. The mixture of organic components (i.e., ECM) and inorganic components (i.e., calcium phosphates) was designed to be "biomimetic" and approximate the molecular composition of native bone with a 1:2 organic/inorganic (wt/wt) component makeup.24 Within the inorganic component, a biphasic mixture of calcium phosphates was used, comprising 20% hydroxyapatite and 80% beta-tricalcium phosphate, which reflects the ratio previously demonstrated to optimize bone formation in vivo.25,26 Last, the material was infused with PDA, as previously described.2123

To make BioMim-PDA, ECM particles were generated from porcine small intestinal submucosa, as previously described.27 Briefly, fresh porcine small intestines were obtained, from which the submucosal layer was mechanically separated. The ECM was then decellularized and sterilized using an aqueous solution of peracetic acid and ethanol. Afterward, the ECM was lyophilized and cryogenically milled to obtain a fine powder of organic components (Supplemental Fig. 1). The ECM powder was then enzymatically digested with pepsin in a dilute HCl solution (pH 2; 10 mg ECM/ml solution) for 24 hours.28 The pH was then adjusted to 7.4 to deactivate pepsin and initiate gelation. Immediately afterward, an 80:20 biphasic mixture of beta-tricalcium phosphate and hydroxyapatite was added. This mixture was stirred for 3 minutes to form a homogeneous mixture of ECM and calcium phosphates that was poured into a mold (2 × 3 inches) and warmed to 37°C. The mixture was then lyophilized, yielding a white, porous biomimetic bone graft material (Supplemental Fig. 1).

The resultant scaffold was then divided into implant-sized pieces that were infused with PDA by immersing them in a dopamine solution (2 mg dopamine HCl/ml 10 mM tris buffer, pH 8.5) with gentle agitation for 5 hours. The PDA-infused grafts were washed with distilled water to remove excess/unattached dopamine molecules and then lyophilized, yielding pieces of BioMim-PDA (Supplemental Fig. 2).

Characterization of BioMim-PDA and a Collagen Sponge by Histology and Scanning Electron Microscopy

Histology and scanning electron microscopy (SEM) were used to characterize the materials properties of BioMim-PDA and the collagen sponge from the Infuse product. For histology, implant-sized pieces of BioMim-PDA and the collagen sponge were dehydrated in ethanol and then embedded in paraffin. Long-cut sections (7 µm thick) were obtained, deparaffinized, and then rehydrated. Staining with H&E, Masson’s trichrome, and von Kossa stains was then performed. Separately, SEM (LEO field emission, ZEISS) was performed (×100 magnification, accelerating voltage 1.0 kV) to investigate the microarchitectures of BioMim-PDA and the collagen sponge.

Preclinical Evaluation Using a Clinically Translatable Rat Model of Spinal Fusion—Direct Comparison With Infuse Bone Graft

To compare the ability of BioMim-PDA to promote bone formation in vivo relative to Infuse, a rat model of L4–5 posterolateral fusion was used,29 with control animals receiving fusion with Infuse. Sixty male Sprague Dawley rats (11–13 weeks of age, approximately 350 g body weight) were randomly assigned to one of six equally sized groups (Table 2). Sample size was calculated a priori using a power analysis for a balanced one-way ANOVA test, with α = 0.05, β = 0.80, and an expected intergroup difference (in bone volume [BV]) of 0.30. Grafts were instilled with rhBMP-2 at three doses—0.2, 2.0, and 20 µg—to evaluate the effects of the interaction of graft type and rhBMP-2 concentration on fusion outcomes. A dose of 0.2 µg rhBMP-2 per side was chosen as the lowest dose, as prior literature reports have suggested that such a dose infused in the collagen sponge would be subtherapeutic and would lead to nonunion (negative control).30 The collagen sponge and rhBMP-2 were obtained from a Large II Kit of Infuse, which was unexpired at the time of use. Aliquots of 0.2, 2.0, and 20 µg rhBMP-2 in 50 µl water were prepared, frozen at −20°C, and stored for later use (< 2 weeks from preparation in all cases). Immediately prior to use, the aliquot was thawed and uniformly distributed onto the collagen sponge or BioMim-PDA. All transfers of rhBMP-2 were performed using ultra-low-binding tubes/pipettes to facilitate maximal transfer of protein. The wetted graft (collagen or BioMim-PDA) was allowed to stand for 15 minutes before implantation, according to the clinical instructions for Infuse (Fig. 1).

TABLE 2.

Study group characteristics (n = 10 rats per group)

Group DesignationBone Graft Substitute MaterialDose of rhBMP-2, µg/Side*
1Infuse collagen sponge0.2
2BioMim-PDA
3Infuse collagen sponge2.0
4BioMim-PDA
5Infuse collagen sponge20
6BioMim-PDA

rhBMP-2 obtained from Infuse contained the additives sucrose, glycine, l-glutamic acid, sodium chloride, and polysorbate 80.

FIG. 1.
FIG. 1.

Comparison of BioMim-PDA to the Infuse collagen sponge. A: Implant-sized pieces of BioMim-PDA (upper row) and collagen sponge (lower row) are shown. B and C: The dry dimensions of all implants were 15 × 5 mm (length × width). D–F: rhBMP-2 (0.2, 2.0, or 20 µg) in aqueous solution was uniformly distributed onto BioMim-PDA or collagen sponge, and the wetted graft was allowed to stand for 15 minutes before implantation; the wetted BioMim-PDA and collagen sponge grafts assumed nearly identical physical dimensions and handleability. Figure is available in color online only.

The model of posterolateral lumbar (L4–5) spinal fusion was performed with an identical procedure in all 60 rats. Briefly, rats were weighed and sedated with a combination of xylazine (5 µg/g rat) and ketamine (70 µg/g rat). The hair on the back of the rat was clipped from the midthoracic spine to the sacrum, and the skin was sterilized with povidone iodine. A midline skin incision over the spinous processes from T10 to the sacrum was then made. The L4–5 transverse processes were exposed bilaterally through paravertebral incisions and decorticated. The bone graft substitute material was then implanted over the decorticated transverse processes (Fig. 2) and kept in place by reapproximation of the overlying paraspinal muscles. The fascia and skin were closed in layers with absorbable suture, and the rat was administered saline (1 ml/100 g rat). The rats were closely monitored postoperatively until fully awake, at which point they were returned to a clean cage. Postoperatively, all rats were housed individually in standard cages and in an identical manner. All rats were euthanized at 8 weeks postoperatively and their spines collected and frozen at −80°C for later analysis.

FIG. 2.
FIG. 2.

Preclinical evaluation using a rat model of posterolateral spinal fusion. The L4–5 transverse processes were exposed bilaterally and decorticated. rhBMP-2–adsorbed collagen sponge (left) or BioMim-PDA (right) was then implanted over the decorticated transverse processes bilaterally; both graft materials were easily moldable around the spine while retaining structural integrity. Figure is available in color online only.

This animal study was approved by the Animal Care and Use Committee of Johns Hopkins University.

Assessment of Spinal Fusion by Microcomputed Tomography and Histology

The spines were analyzed using high-resolution microcomputed tomography (µCT) and histology. Scans were performed using a SkyScan 1275 (Bruker) with 30-µm slice thickness. Successful fusion was defined from the µCT imaging as continuous bridging bone from L4 to L5, bilaterally. In addition, quantitative µCT analysis of the fusion masses was performed using CTAn (version 1.18.8.0, Bruker) to evaluate for BV, as well as three parameters of bone quality: percent BV (%BV; BV divided by fusion mass volume), trabecular number (TN; number of trabeculae per micrometer [µm]), and trabecular separation (TS; distance [µm] between trabeculae). The fusion mass was defined as the volume of space lateral to the L4 and L5 vertebral bodies, including the transverse processes. The µCT analysis was performed in blinded fashion. Additionally, 3D reconstructions of the µCT scans were performed using CTVox (version 3.3.0, Bruker) to macroscopically visualize the fusion masses.

After the µCT analysis was performed, a representative sample from each group was prepared for histological analysis to evaluate the formation of bone, cartilage, and fibrous tissue. Samples were fixed in 4% paraformaldehyde solution, decalcified in RDO Rapid Decalcifier (Apex Engineering), dehydrated in ethanol, and then embedded in paraffin. Coronal sections were cut at a thickness of 7 µm, deparaffinized, rehydrated, and then stained with H&E.

Statistical Analysis

The means and standard deviations of BV, %BV, TN, and TS for each group were calculated. To compare between groups, we performed one-way ANOVA with post hoc Tukey honestly significant difference (HSD) testing. All statistical analysis was performed using R version 3.6.2 (R Foundation for Statistical Computing), with statistical significance defined as p < 0.05. We hypothesized that using rhBMP-2 with BioMim-PDA would lead to greater BV and bone quality at each of the three doses of rhBMP-2 versus using the collagen sponge.

Results

Characterization of BioMim-PDA and a Collagen Sponge

Comparative histological stains of BioMim-PDA and the collagen sponge using H&E, Masson’s trichrome, and von Kossa stains are shown in Supplemental Fig. 3. As seen with the H&E and Masson’s trichrome stains, both materials are porous and acellular and comprise homogeneously distributed collagens. However, as seen in the von Kossa stains, BioMim-PDA, unlike the collagen sponge supplied with Infuse bone graft, also comprises homogeneously distributed calcium phosphates. Similarly, SEM imaging illustrates the porous, homogeneous, and fibrillar architecture of both materials (Supplemental Fig. 4).

Preclinical Evaluation Using the Rat Model of Spinal Fusion—Direct Comparison of BioMim-PDA and the Collagen Sponge With Different Concentrations of rhBMP-2

All 60 rats undergoing surgery survived until the end of the study (8 weeks postoperatively) and were evaluated. There were no wound site infections, instances of wound dehiscence, or other postoperative complications requiring intervention.

The fusion rate was 100% in all groups except group 1 (collagen sponge + 0.2 µg rhBMP-2; 70%) and group 4 (BioMim-PDA + 2.0 µg rhBMP-2; 90%). The one failed fusion in group 4 was attributed to an intraoperative complication leading to excessive paraspinal bleeding and poor graft placement on one side, which was documented at the time of surgery. Unilateral fusion was observed on the unaffected side. There were no other instances of intraoperative complications.

A summary of the quantitative µCT analysis is shown in Table 3 and Supplemental Fig. 5. Significantly greater BV, %BV, and TN, as well as significantly smaller TS, were observed with the use of BioMim-PDA than with the use of the collagen sponge at each of the three concentrations of rhBMP-2. Furthermore, use of BioMim-PDA with 0.2 µg rhBMP-2 led to significantly greater BV (125 vs 101 µm3), %BV (32.6% vs 23.9%), and TN (2.0 vs 1.6/µm), as well as significantly smaller TS (0.39 vs 0.63 µm), compared with the use of the collagen sponge with 2.0 µg rhBMP-2. Similar observations were noted when comparing the use of BioMim-PDA with 2.0 µg rhBMP-2 to the use of the collagen sponge with 20 µg rhBMP-2.

TABLE 3.

Summary of quantitative µCT analysis

VariablerhBMP-2, µg/Side*F-Testp ValueTukey HSD
0.22.020
BV, µm3
 Collagen sponge79.4 ± 11.7101.3 ± 10.4106.6 ± 14.857.9<0.001All, except groups 2–5 & 3–5
 BioMim-PDA125.1 ± 20.4152.5 ± 16.4182.8 ± 17.8
BV, %
 Collagen sponge27.9 ± 2.323.9 ± 3.015.0 ± 3.236.4<0.001All, except groups 1–3, 1–4, 2–4, & 3–6
 BioMim-PDA32.6 ± 2.328.6 ± 3.422.8 ± 4.4
TN, 1/µm
 Collagen sponge1.723 ± 0.1371.585 ± 0.2011.033 ± 0.20034.9<0.001All, except groups 1–3, 1–4, 2–4, & 3–6
 BioMim-PDA2.005 ± 0.1301.836 ± 0.1831.483 ± 0.214
TS, µm
 Collagen sponge0.489 ± 0.0410.631 ± 0.0901.024 ± 0.14557.8<0.001All, except groups 1–2, 1–4, 2–4, & 3–6
 BioMim-PDA0.392 ± 0.0320.497 ± 0.0870.727 ± 0.119

Group 1 = collagen sponge + 0.2 µg rhBMP-2/side; group 2 = BioMim-PDA + 0.2 µg rhBMP-2/side; group 3 = collagen sponge + 2.0 µg rhBMP-2/side; group 4 = BioMim-PDA + 2.0 µg rhBMP-2/side; group 5 = collagen sponge + 20 µg rhBMP-2/side; group 6 = BioMim-PDA + 20 µg rhBMP-2/side.

Values are presented as mean ± SD.

Significant differences (p < 0.05) between groups.

Representative 3D reconstructions of the µCT data for each group are shown in Fig. 3. Additionally, representative µCT slices and corresponding histological images at the same level in the coronal plane are presented in Fig. 4. Spinal fusions are shown for groups 2–6, and a unilateral nonunion comprising fibrous tissue is shown for group 1. As seen in both the radiographic and histological imaging, increases in the amount of rhBMP-2 led to decreases in %BV and TN, as well as an increase in TS, for both graft material types (Table 3, Figs. 3 and 4).

FIG. 3.
FIG. 3.

Representative 3D reconstructions of the µCT data. Significantly greater BV and bone quality were observed with BioMim-PDA than the collagen sponge at each of the three concentrations of rhBMP-2 evaluated: 0.2 µg rhBMP-2 used with the collagen sponge (A) versus BioMim-PDA (B), 2.0 µg rhBMP-2 used with the collagen sponge (C) versus BioMim-PDA (D), and 20 µg rhBMP-2 used with the collagen sponge (E) versus BioMim-PDA (F). In addition, use of BioMim-PDA with 0.2 µg rhBMP-2 (B) led to significantly greater BV and bone quality compared with use of the collagen sponge with 2.0 µg rhBMP-2 (C). Similar observations were noted when comparing the use of BioMim-PDA with 2.0 µg rhBMP-2 (D) to the use of the collagen sponge with 20 µg rhBMP-2 (E).

FIG. 4.
FIG. 4.

Representative µCT slices and corresponding H&E histological images at the same level (coronal plane). A: Example of a unilateral fibrous nonunion (right side) following use of the collagen sponge and 0.2 µg rhBMP-2. B–F: For the remaining groups, spinal fusions are shown, defined as continuous bridging bone from L4 to L5, bilaterally. Increases in the amount of rhBMP-2 led to increases in BV for both graft material types. Figure is available in color online only.

Discussion

Here we report the creation and preclinical evaluation of BioMim-PDA, a novel bone graft substitute material that was specifically designed to overcome the limitations of the Infuse bone graft material (Table 1). We designed BioMim-PDA to have materials properties similar to those of the collagen sponge of the Infuse product and to be used in an identical manner (e.g., absorb fluid, be moldable about the site of grafting, and fit into an interbody device). The BioMim-PDA matrix comprises a 1:2 mixture of organic/inorganic components instilled with PDA, which enables a controlled release of rhBMP-2 and thus facilitates a more physiological delivery of rhBMP-2 relative to that seen from sponges comprising collagen alone. By use of a clinically translatable rat model of spinal fusion, rhBMP-2–infused BioMim-PDA grafts achieved BV and bone quality superior to what was seen with the use of collagen sponges infused with a 10-fold higher concentration of rhBMP-2 (Table 4). Consequently, our results suggest that by employing the more physiological BioMim-PDA scaffold, rhBMP-2–supplemented fusions could achieve successful radiographic fusions at significantly lower rhBMP-2 concentrations, with concordantly lower biological reagent costs and lower rates of rhBMP-2–related side effects.

TABLE 4.

Summary of composition ingredients, instructions for use, and efficacy of the collagen sponge and BioMim-PDA used with rhBMP-2 for bone grafting

Bone Graft Substitute MaterialScaffold CompositionInstructions for UseEfficacy*
Collagen sponge + rhBMP-2 (Infuse bone graft)Bovine type I collagenDistribute rhBMP-2 onto the collagen sponge, wait 15 mins, then implantRef
BioMim-PDA + rhBMP-2Porcine small intestinal submucosa, calcium phosphates, & PDASame as above (replacing the collagen sponge w/ BioMim-PDA)Greater bone formation w/ 1/10 the dose of rhBMP-2 used w/ collagen sponge

Evaluated using a rat model of spinal fusion.

Infuse bone graft consists of a type I collagen sponge and an aqueous solution of rhBMP-2 and excipients (including sucrose, glycine, l-glutamic acid, sodium chloride, and polysorbate 80).

rhBMP-2–adsorbed collagen sponges (e.g., Infuse) have proven enormously successful as bone graft substitute materials; however, their efficacy suffers from the use of a simple collagen sponge, which has minimal inherent osteoconductive/osteoinductive properties and an extremely limited ability to control the release of adsorbed rhBMP-2.31,32 The strength of these graft substitutes stems from the incorporation of rhBMP-2, which remains among the most potent osteoinductive agents known.33 Prior studies have demonstrated that by simply combining calcium phosphates with the collagen material, a graft substitute can be produced with materials properties similar to those of a conventional collagen sponge, but with the inherent osteoinductivity of calcium phosphates. In vitro studies have shown such grafts to increase cell adhesion and viability, osteogenic gene expression, vascular density, and bone formation compared with the use of either collagen or calcium phosphates alone.3438

However, these previous calcium phosphate–collagen composites still lack many of the essential osteoconductive and osteoinductive properties of native tissue. A superior alternative is offered by pro-regenerative ECMs, which offer 3D microarchitecture for mechanical support and signaling, as well as a reservoir of biochemical cues to orchestrate physiological tissue generation.39 To this end, products based on pro-regenerative ECMs, like small intestinal submucosa, have been FDA approved and commercialized for the reconstruction of soft-tissue defects.40,41 However, these products have yet to be translated to clinical applications of bone tissue engineering. The graft described herein represents what is to our knowledge the first such material. Last, the use of PDA—a mussel-inspired bioadhesive—provides an additional improvement upon existing graft materials that employ exogenously applied growth factors without a drug delivery strategy.21,42 PDA facilitates a controlled release of rhBMP-2 from the graft material, unlike the collagen sponge, which is associated with an initial burst release of supraphysiologic levels of rhBMP-2, followed by a significant diminution in local rhBMP-2 levels.31,32 The application of PDA to graft materials has been shown to reduce the initial burst release by more than half relative to nonactivated control materials, as well as provide sustained growth factor release over weeks-long time frames.23,43,44 To our knowledge, the present work is the first to describe the development of a graft substitute incorporating all these technologies—namely, a pro-regenerative ECM-based biomimetic substrate instilled with PDA and infused with rhBMP-2.

Although further investigation is warranted, replacing collagen with BioMim-PDA in rhBMP-2–supplemented bone grafting procedures offers significant potential cost savings, which are driven by theoretical reductions in the amount of rhBMP-2 needed. Broad estimates of costs can be made by considering the costs associated with producing/procuring the two components of the grafts: 1) the scaffold and 2) the growth factor. In terms of the scaffold, both the collagen sponge and BioMim-PDA are relatively inexpensive: pure type I collagen sponges as used in Infuse are sold commercially (e.g., HeliPlug, HeliCote, and Helistat [Integra LifeSciences]) for tens of dollars per unit (e.g., $20), whereas the cost to produce BioMim-PDA is slightly higher. In contrast, growth factors are expensive: rhBMP-2 sells commercially for approximately $7500/mg (e.g., R&D Systems, Thermo Fisher Scientific), whereas the amount of rhBMP-2 used in Infuse ranges from 1.05 mg (XX Small Kit) to 12 mg (Large II Kit). To this end, even with a moderate increase in cost to produce BioMim-PDA relative to collagen, the order-of-magnitude decrease in the amount of rhBMP-2 needed for fusion potentially translates to thousands of dollars in cost savings per patient.

There are several limitations to the present study, the foremost of which is its animal design. We followed the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines,45 as well as utilized an established preclinical model of spinal fusion, to strengthen the quality of the present study and the conclusions that can be drawn from it. In addition, we did not evaluate the release kinetics of rhBMP-2 from the collagen sponge or BioMim-PDA, which may have been useful for optimizing physiological drug delivery parameters but would have amounted to a separate study. Additionally, the operative investigator could not be blinded to graft type because of clear visual differences between BioMim-PDA and the collagen sponge. Further, we did not evaluate the range of complications associated with the use of Infuse in this study. Despite these limitations, our results provide initial evidence that rhBMP-2–supplemented fusions using BioMim-PDA are safe and more cost-effective than those using collagen. Our future work will focus on optimizing the composition of the BioMim-PDA scaffold for bone grafting and fusions, including investigating versions with additives (e.g., small molecules), as well as investigating rhBMP-2 release kinetics. Our ultimate goal in the present work was to make bone graft substitute materials that are safe, efficacious, and cost-effective for any patient requiring a bone graft or fusion.

Conclusions

BioMim-PDA—a PDA-infused, porous, homogeneously dispersed solid mixture of ECM and calcium phosphates—is a promising novel bone graft substitute material. In the present study, performed in a rat model of spinal fusion, this new graft material resulted in superior BV and bone quality than was seen in collagen sponges infused with a 10-fold higher concentration of rhBMP-2. Additional work is needed to evaluate the merits of BioMim-PDA more fully in conjunction with rhBMP-2 as a safe and efficacious bone graft substitute.

Acknowledgments

We thank the Johns Hopkins University School of Medicine Microscope Core Facility for microscopy services. This work was directly supported by the National Institute on Aging, the National Institute of General Medical Sciences, the National Cancer Institute Intramural Research Program, and the Gordon and Marilyn Macklin Foundation.

Disclosures

Dr. Cottrill reported grants from the National Institute on Aging and National Institute of General Medical Sciences during the conduct of the study and a patent pending for intellectual property related to biological scaffolds. Dr. Sciubba reported personal fees from DePuy Synthes, Medtronic, Stryker, and Baxter outside the submitted work. Dr. Theodore reported personal fees from Globus Medical outside the submitted work. Dr. Witham reported grants from the Gordon and Marilyn Macklin Foundation during the conduct of the study and a patent pending for intellectual property related to biological scaffolds.

Author Contributions

Conception and design: Cottrill, Wolf, Lambrechts, Sciubba, Witham. Acquisition of data: Cottrill, Dirckx, Perdomo-Pantoja, Rajkovic, Lin, Witham. Analysis and interpretation of data: Cottrill, Wolf, Dirckx, Mageau, Theodore, Witham. Drafting the article: Cottrill, Pennington, Witham. Critically revising the article: Cottrill, Pennington, Ehresman, Perdomo-Pantoja, Rajkovic, Maestas, Theodore, Elisseeff, Sciubba, Witham. Reviewed submitted version of manuscript: Cottrill, Pennington, Wolf, Ehresman, Perdomo-Pantoja, Rajkovic, Maestas, Mageau, Sciubba, Theodore, Elisseeff, Witham. Approved the final version of the manuscript on behalf of all authors: Cottrill. Statistical analysis: Cottrill. Administrative/technical/material support: Cottrill, Perdomo-Pantoja, Lin, Maestas, Mageau, Stewart, Theodore, Elisseeff, Witham. Study supervision: Sciubba, Theodore, Elisseeff, Witham.

Supplemental Information

Online-Only Content

Supplemental material is available with the online version of the article.

Previous Presentations

Portions of this work were previously presented in abstract form at the 2021 Annual Meeting of the Lumbar Spine Research Society, April 8–10, 2021 (virtual).

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    Cottrill E, Premananthan C, Pennington Z, et al. Radiographic and clinical outcomes of silicate-substituted calcium phosphate (SiCaP) bone grafts in spinal fusion: systematic review and meta-analysis. J Clin Neurosci. 2020;81:353366.

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    Perdomo-Pantoja A, Holmes C, Cottrill E, et al. Comparison of freshly isolated adipose tissue-derived stromal vascular fraction and bone marrow cells in a posterolateral lumbar spinal fusion model. Spine (Phila Pa 1976). 2021;46(10):631637.

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Supplementary Materials

  • Collapse
  • Expand
Figure from Vedantam et al. (pp 28–39).
  • FIG. 1.

    Comparison of BioMim-PDA to the Infuse collagen sponge. A: Implant-sized pieces of BioMim-PDA (upper row) and collagen sponge (lower row) are shown. B and C: The dry dimensions of all implants were 15 × 5 mm (length × width). D–F: rhBMP-2 (0.2, 2.0, or 20 µg) in aqueous solution was uniformly distributed onto BioMim-PDA or collagen sponge, and the wetted graft was allowed to stand for 15 minutes before implantation; the wetted BioMim-PDA and collagen sponge grafts assumed nearly identical physical dimensions and handleability. Figure is available in color online only.

  • FIG. 2.

    Preclinical evaluation using a rat model of posterolateral spinal fusion. The L4–5 transverse processes were exposed bilaterally and decorticated. rhBMP-2–adsorbed collagen sponge (left) or BioMim-PDA (right) was then implanted over the decorticated transverse processes bilaterally; both graft materials were easily moldable around the spine while retaining structural integrity. Figure is available in color online only.

  • FIG. 3.

    Representative 3D reconstructions of the µCT data. Significantly greater BV and bone quality were observed with BioMim-PDA than the collagen sponge at each of the three concentrations of rhBMP-2 evaluated: 0.2 µg rhBMP-2 used with the collagen sponge (A) versus BioMim-PDA (B), 2.0 µg rhBMP-2 used with the collagen sponge (C) versus BioMim-PDA (D), and 20 µg rhBMP-2 used with the collagen sponge (E) versus BioMim-PDA (F). In addition, use of BioMim-PDA with 0.2 µg rhBMP-2 (B) led to significantly greater BV and bone quality compared with use of the collagen sponge with 2.0 µg rhBMP-2 (C). Similar observations were noted when comparing the use of BioMim-PDA with 2.0 µg rhBMP-2 (D) to the use of the collagen sponge with 20 µg rhBMP-2 (E).

  • FIG. 4.

    Representative µCT slices and corresponding H&E histological images at the same level (coronal plane). A: Example of a unilateral fibrous nonunion (right side) following use of the collagen sponge and 0.2 µg rhBMP-2. B–F: For the remaining groups, spinal fusions are shown, defined as continuous bridging bone from L4 to L5, bilaterally. Increases in the amount of rhBMP-2 led to increases in BV for both graft material types. Figure is available in color online only.

  • 1

    Martin BI, Mirza SK, Spina N, Spiker WR, Lawrence B, Brodke DS. Trends in lumbar fusion procedure rates and associated hospital costs for degenerative spinal diseases in the United States, 2004 to 2015. Spine (Phila Pa 1976). 2019;44(5):369376.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 2

    Rajaee SS, Bae HW, Kanim LE, Delamarter RB. Spinal fusion in the United States: analysis of trends from 1998 to 2008. Spine (Phila Pa 1976). 2012;37(1):6776.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 3

    McDermott K, Freeman W, Elixhauser A. HCUP Statistical Brief #233: Overview of Operating Room Procedures During Inpatient Stays in U.S. Hospitals, 2014.Agency for Healthcare Research and Quality;2017:118.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 4

    Tsutsumimoto T, Shimogata M, Yoshimura Y, Misawa H. Union versus nonunion after posterolateral lumbar fusion: a comparison of long-term surgical outcomes in patients with degenerative lumbar spondylolisthesis. Eur Spine J. 2008;17(8):11071112.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 5

    Noshchenko A, Lindley EM, Burger EL, Cain CM, Patel VV. What is the clinical relevance of radiographic nonunion after single-level lumbar interbody arthrodesis in degenerative disc disease? A meta-analysis of the YODA Project database. Spine (Phila Pa 1976). 2016;41(1):917.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 6

    McAnany SJ, Baird EO, Overley SC, Kim JS, Qureshi SA, Anderson PA. A meta-analysis of the clinical and fusion results following treatment of symptomatic cervical pseudarthrosis. Global Spine J. 2015;5(2):148155.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 7

    Chun DS, Baker KC, Hsu WK. Lumbar pseudarthrosis: a review of current diagnosis and treatment. Neurosurg Focus. 2015;39(4):E10.

  • 8

    Cottrill E, Pennington Z, Ahmed AK, et al. The effect of electrical stimulation therapies on spinal fusion: a cross-disciplinary systematic review and meta-analysis of the preclinical and clinical data. J Neurosurg Spine. 2020;32(1):106126.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 9

    Cottrill E, Downey M, Pennington Z, et al. Low-intensity pulsed ultrasound as a potential adjuvant therapy to promote spinal fusion: systematic review and meta-analysis of the available data. J Ultrasound Med. 2021;40(10):20052017.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 10

    Cottrill E, Ahmed AK, Lessing N, et al. Investigational growth factors utilized in animal models of spinal fusion: systematic review. World J Orthop. 2019;10(4):176191.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 11

    Cottrill E, Lazzari J, Pennington Z, et al. Oxysterols as promising small molecules for bone tissue engineering: systematic review. World J Orthop. 2020;11(7):328344.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 12

    Cottrill E, Pennington Z, Lankipalle N, et al. The effect of bioactive glasses on spinal fusion: a cross-disciplinary systematic review and meta-analysis of the preclinical and clinical data. J Clin Neurosci. 2020;78:3446.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 13

    Cottrill E, Premananthan C, Pennington Z, et al. Radiographic and clinical outcomes of silicate-substituted calcium phosphate (SiCaP) bone grafts in spinal fusion: systematic review and meta-analysis. J Clin Neurosci. 2020;81:353366.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 14

    Carragee EJ, Hurwitz EL, Weiner BK. A critical review of recombinant human bone morphogenetic protein-2 trials in spinal surgery: emerging safety concerns and lessons learned. Spine J. 2011;11(6):471491.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 15

    McKay WF, Peckham SM, Badura JM. A comprehensive clinical review of recombinant human bone morphogenetic protein-2 (INFUSE Bone Graft). Int Orthop. 2007;31(6):729734.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 16

    James AW, LaChaud G, Shen J, et al. A review of the clinical side effects of bone morphogenetic protein-2. Tissue Eng Part B Rev. 2016;22(4):284297.

  • 17

    Hustedt JW, Blizzard DJ. The controversy surrounding bone morphogenetic proteins in the spine: a review of current research. Yale J Biol Med. 2014;87(4):549561.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 18

    El Bialy I, Jiskoot W, Reza Nejadnik M. Formulation, delivery and stability of bone morphogenetic proteins for effective bone regeneration. Pharm Res. 2017;34(6):11521170.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 19

    Gamradt SC, Lieberman JR. Genetic modification of stem cells to enhance bone repair. Ann Biomed Eng. 2004;32(1):136147.

  • 20

    Zara JN, Siu RK, Zhang X, et al. High doses of bone morphogenetic protein 2 induce structurally abnormal bone and inflammation in vivo. Tissue Eng Part A. 2011;17(9-10):13891399.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 21

    Lee H, Dellatore SM, Miller WM, Messersmith PB. Mussel-inspired surface chemistry for multifunctional coatings. Science. 2007;318(5849):426430.

  • 22

    Lee SJ, Lee D, Yoon TR, et al. Surface modification of 3D-printed porous scaffolds via mussel-inspired polydopamine and effective immobilization of rhBMP-2 to promote osteogenic differentiation for bone tissue engineering. Acta Biomater. 2016;40:182191.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 23

    Sun H, Dong J, Wang Y, et al. Polydopamine-coated poly(l-lactide) nanofibers with controlled release of VEGF and BMP-2 as a regenerative periosteum. ACS Biomater Sci Eng. 2021;7(10):48834897.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 24

    Feng X. Chemical and biochemical basis of cell-bone matrix interaction in health and disease. Curr Chem Biol. 2009;3(2):189196.

  • 25

    Arinzeh TL, Tran T, Mcalary J, Daculsi G. A comparative study of biphasic calcium phosphate ceramics for human mesenchymal stem-cell-induced bone formation. Biomaterials. 2005;26(17):36313638.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 26

    van Esterik FA, Zandieh-Doulabi B, Kleverlaan CJ, Klein-Nulend J. Enhanced osteogenic and vasculogenic differentiation potential of human adipose stem cells on biphasic calcium phosphate scaffolds in fibrin gels. Stem Cells Int. 2016;2016:1934270.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 27

    Badylak SF, Freytes DO, Gilbert TW. Extracellular matrix as a biological scaffold material: structure and function. Acta Biomater. 2009;5(1):113.

  • 28

    Freytes DO, Martin J, Velankar SS, Lee AS, Badylak SF. Preparation and rheological characterization of a gel form of the porcine urinary bladder matrix. Biomaterials. 2008;29(11):16301637.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 29

    Perdomo-Pantoja A, Holmes C, Cottrill E, et al. Comparison of freshly isolated adipose tissue-derived stromal vascular fraction and bone marrow cells in a posterolateral lumbar spinal fusion model. Spine (Phila Pa 1976). 2021;46(10):631637.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 30

    Bae HW, Zhao L, Kanim LE, Wong P, Marshall D, Delamarter RB. Bone marrow enhances the performance of rhBMP-2 in spinal fusion: a rodent model. J Bone Joint Surg Am. 2013;95(4):338347.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 31

    Geiger M, Li RH, Friess W. Collagen sponges for bone regeneration with rhBMP-2. Adv Drug Deliv Rev. 2003;55(12):16131629.

  • 32

    Li RH, Wozney JM. Delivering on the promise of bone morphogenetic proteins. Trends Biotechnol. 2001;19(7):255265.

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